The overall objective of this proposal is to determine at the molecular level how changes in intracellular pH regulate cell migration. In the previous two funding cycles we established that H+ efflux by the Na-H exchanger is necessary for directed cell migration. We found that NHE1 is anchored to actin filaments, which localizes NHE1 at the distal margin of membrane protrusions. We showed a leading-edge H+ efflux by NHE1 in mammalian fibroblasts and in Dictyostelium cells is necessary for three stages in cell migration: polarity, actin filament assembly driving membrane protrusion, and cell-substrate adhesion remodeling. We also began studying pH sensors, or proteins with activities or ligand-binding affinities that are regulated by physiological changes in pH. Intracellular pH sensors predicted to mediate NHE1-dependent cell migration were examined by computational modeling, NMR, and functional studies. The current proposal investigates the hypothesis that pH sensors play critical roles in regulating cell migration. Our studies bridge cell biology and structural biology to determine at the molecular level the regulation and function of pH sensors in cell polarity, actin-dependent membrane protrusion, and cell adhesion. In Aim 1 we identify components of the positive feedback loop between NHE1 and Cdc42 required for fibroblast cell polarity. We will ask how NHE1 stimulates Cdc42 activity by testing the prediction that GEFs mediating NHE1-dependent activation of Cdc42 are pH sensors with pH- dependent PI(4,5)P2 binding regulated by a histidine switch. We also will ask how Cdc42-GTP stimulates NHE1 activity by testing the prediction that increased phosphorylation of NHE1 is necessary for its activation by Cdc42-GTP and for cell polarity. In Aim 2 we identify mechanisms regulating the biphasic kinetics of actin filament assembly for membrane protrusion. We will ask how cofilin and actin-interacting protein 1 (Aip1) function in NHE1-dependent actin dynamics by testing the prediction that changes in pH regulate Aip1 structure and binding to cofilin to confer net actin filament assembly and cell migration. New findings that phosphorylation of the Arp2 subunit of the Arp2/3 complex is necessary for nucleating actin filaments provides the rationale to ask how pArp2 regulates actin kinetics in response to migratory cues. We will test whether regulated phosphorylation of Arp2 has distinct functions in actin dynamics and membrane protrusion in motile Dictyostelium cells and mammalian fibroblasts by using biochemical assays and high resolution imaging of live cells. In Aim 3 we determine the role of NHE1-regulated focal adhesion proteins in remodeling cell-substrate adhesions and in directed migration of fibroblasts. We will ask how autophosphorylation of FAK is NHE1- dependent by testing the prediction that protonation of histidine residues in the FERM domain of FAK sterically inhibits autophosphorylation. Studies in cells will test the function of a mutant pH-insensitive FAK in focal adhesion dynamics and cell migration. We also will ask the functional significance of 1-actinin binding to NHE1 by using NHE with mutations in the 1-actinin binding site. PUBLIC HEALTH RELEVANCE: Cell migration plays a critical role in a number of physiological processes, including development, axonal guidance, immune responses, and wound healing, and in pathologies, including atherosclerosis and the metastasis and invasion of tumor cells. Because intracellular pH is an evolutionarily-conserved regulator of directed cell migration, identifying pH-regulated cell migration at the molecular level will advance our understanding of how migratory stages are controlled and integrated and can be inhibited to restrict pathologies.